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Genomics Training A Cooperative Endeavor between USEPA Region 5 -and- USEPA Office of Research and Development Adam Biales NERL Postdoctoral Fellow Overview DNA RNA Protein PCR The Central Dogma Central dogma of molecular biology states that DNA carries the genetic information which is transcribed to RNA and subsequently translated to protein DNA RNA Protein The Central Dogma DNA RNA Protein Overview of DNA DNA stores information – Blue print – – – – 1-5 billion base pairs in length 2 meters in length Very complex Lots more DNA than genes Non-coding – Repetitive (microsatellites, SINES, etc.) – Introns – Structural – telomeres, centromeres Protein coding ≈ 1% – Complexity becoming clear 1970 ≈ 100 million genes 2002 ≈ 30,000 genes Mitochondrial vs. Nuclear Nucleosome Chromosomes Overview of DNA DNA stores information – Blue print – Very complex – Lots more DNA than genes Non-coding – Repetitive (microsatellites, SINES, etc.) – Introns – Structural – telomeres, centromeres Protein coding ≈ 1% – Complexity becoming clear 1970 ≈ 100 million genes 2002 ≈ 30,000 genes Mitochondrial vs. Nuclear DNA structure DNA is a polymer of nucleotides – Purines (Adenine,Guanine) – Pyrimidines (Cytosine,Thymine) Two strands twist around each other (double helix) – Bases of the two strands face each other – Forms base pairs – A pairs with T, G pairs with C Nucleotide DNA structure DNA is a polymer of nucleotides – Purines (Adenine,Guanine) – Pyrimidines (Cytosine,Thymine) Two strands twist around each other (double helix) – Bases of the two strands face each other – Forms base pairs – A pairs with T, G pairs with C The Central Dogma DNA RNA Protein Overview of RNA Different types – mRNA, tRNA, rRNA Carries message from DNA to ribosome – protein Often quick turn over Highly regulated All functions not understood RNA structure – Single stranded – DNA and RNA have structural differences Pentoses (five-carbon sugar component) – In DNA, the pentose is deoxyribose – In RNA, it is ribose Nitrogenous bases – thymine in DNA, uracil in RNA RNA Structure The Central Dogma DNA RNA Protein Overview of Proteins Made from amino acids strung together Form regular 3-D structures Structure dictates function Subunits add specificity to function Turned on and off by chemical modifications Levels of Protein Structure Overview of Proteins Made from amino acids strung together Form regular 3-D structures Structure dictates function Subunits add specificity to function Turned on and off by chemical modifications Proteins Proteins are used for a variety of functions in the cell: – – – – – Structural support Metabolism Motion Defense DNA replication, RNA synthesis, etc. – And many more functions DNA Polymerase PCR Polymerase Chain Reaction - PCR An in vitro method for replicating DNA Makes billions of copies of a DNA template in a few hours Requires only a tiny amount of starting material Highly specific: primers determine the sequence to be amplified PCR reaction components Template DNA strand Nucleotides (dNTPS) Heat stable DNA Polymerase – Taq – Same enzyme that replicates DNA in your cells – Isolated from hotsprings in Yellowstone Primers (oligos) – 20ish bp Primer specificity 4 nucleotides, 3.2 x 109 base pairs in the human genome… An oligo of 10 bases in length = 410 (an exact sequence match expected to occur ~ every 1 million bases) …expected to be represented in the genome 3,200 times An oligo of 10 bases in length = 415 (an exact sequence match expected to occur ~ every 1 billion bases) …expected to be represented in the genome 3 times An oligo of 10 bases in length = 420 (an exact sequence match expected to occur ~ every 1 trillion bases) …expected to be represented in the genome…well you get the idea PCR Amplification Cycles Copies 1 2 2 4 4 16 10 1,024 15 32,768 20 1,048,576 25 33,554,432 30 1,073,741,824 Expression Based Technology Expression Based Technology Immediate responses on cellular level Biological information Can be specific for a given stressor Rapid Regulation Needs to be heavily regulated Appropriate cell activation If not regulated leads to pathology – Cancers Cell-cycle Signaling Occurs on each level: – DNA, RNA, Protein Gene Structure Promoter Cis element 1000 - 250000 bp Regulation DNA Gene Structure Promoter Cis element 1000 - 250000 bp GENE Transcription/RNA processing Intron Exon Intron Exon 5’ Intron Exon 3’ mRNA AAAAAAN250 The Central Dogma Reverse Transcriptase cDNA DNA RNA Protein QPCR Realtime quantitative PCR – Advantages Quantitative Sensitive Reproducible Minimal tissue requirements Rapid results “Real-Time” visualization of amplified products Dynamic range 107 Able to QA 100 Highly reproducible across entire range 12 replicates each DCt < 0.50 cycles Microarrays Monitor changes across 1000s of genes in 1 experiment – – – – Available for rainbow trout Zebrafish FHM – 2006 Daphnia Signatures – 1 set of genes = 1 chemical Systems biology Information about mechanism – Cluster analysis Control (reference sample) Treatment (ie. effluent) Isolate Tissue RNA RT & label Isolate Tissue RNA RT & label 1 1 1 3 2 1 2 2 3 1 2 Microarray 1 Message present only in treatment Message present only in control Message present at equal levels in both treatment and control Adapted from H. Hamadeh and C. Afshari, American Scientist 88:508-515 Genes Expression Comparison between High and Low Grade Cancer Lapointe, Jacques et al. (2004) Proc. Natl. Acad. Sci. USA 101, 811-816 Proteomics Study of protein populations of one cell versus another Similar to microarrays – global changes Protein data more biologically relevant – Higher biological level – Less variable 2-D gel electrophoresis – Non-directed, no assumptions – Can see modifications 2-D Gel Electrophoresis Control Treatment The Exposure-Effect Hierarchy Biological Level Community Biological Effect Bio-assessment + Community Assessment Population Population Decline, Adaptation Population DNA Analyses Organism Toxicity Testing Tissue/Organ Histopathology Cellular Cell death/Mitosis/Activation Subcellular Molecular/RNA/Protein Changes Current Molecular Assays Vitellogenin – Egg protein – only females in breeding season – Males turn it on when exposed to estrogen – All kinds of fish Fathead, trout, bass, gudgeon, shiners, roach, flounder – Can be used as an indicator to estrogen exposure Estrogens Estradiol, estrone, EE2 Sources – Farm runoff – Contraceptives Environmental levels - EE2 – – – – Surface water 0.05 – 30.8 ng/L EE2 Sewage effluents 0.05 – 62.0 ng/L EE2 LOEC Vg protein 0.1 ng/L LOEC sex interchange 0.6 ng/L Can have additive effects Potent responses at low levels – (4 ng/L EE2) Estrogenic Effects Male fish with ovatestes Decreased male fitness Decreased size/physical abnormalities Decreased reproductive success (.32 ng/L) – NO EGGS PRODUCED at 3.5 ng/L Skewed sex ratios – 0 males with secondary sex characters – 3.5 ng/L May have long-term effect on reproductive success – 50% reduction in reproductive success 29 days after exposure – 5 months following treatment – decreased fertility Issues Fish swim – Not sitting by effluent for whole life – Level of real world exposure Linkage between biological levels not completely established Why are there any fish at all? Needs field testing Mixtures Can we use gene expression as a metric in bioassessment ELA Located in northwestern Ontario approximately 250 km east of Winnipeg and 50 km east-southeast of Kenora. K. Kidd ELA Objective: To study the long term effects of xenoestrogens on wild fish population Dose lake with 4-6 ng/L EE2 or not for 3 continuous years (2001-03) Measure Vg expression in wild fish, deployed fish, laboratory, embryo/larval exposures Water chemistry Sediment elutriate exposure in fry Other biological measurements – aggression, mortality ELA results Male FHM had elevated Vg expression from 24 hours until October (last sample of year) Females had elevated Vg levels past end of breeding season Male pearl dace also exhibited high Vg levels In fall of second year no age 0 fish found Histopathology – liver hypertrohpy – fibrotic sperm ducts Ohio River Collaboration with Ohio River Valley Water Sanitation Commission ORSANCO Vg – Sewage Treatment Plants Objective: Create Vg assay and identify the number of species and geographic extent effected by estrogenic compounds found in effluents 3 sampling sites – Region 3 – Wheeling, ALCOSAN, Parkersburg Downstream – proximal Downstream – distal Upstream – reference Multiple species Multiple exposures – lab, deployment – high & low flow Chemical analysis Histopath analysis Sex Ratios Questions – Are male fish in reference sites producing Vg? – If fish aren’t stuck at effluent is there an effect? Percentage of sites within a pool with failing IBI scores Assessment of Ohio River Pools, 2004 100 90 80 70 60 50 40 30 20 10 303d list 40% 41.4% 25.5% 26.4% 25% New C Racine Mark15 Mark29 Myers Ohio River - EDC Using probabilistic sampling to determine extent of exposure in a pool of the Ohio River – Fish localized to a given pool - Dams – 15 probabilistic sites – Exposure differences – ecology Multiple species – Bottom feeders – Water column – Sex ratios – Vg expression – Result: does expression aid in identifying causes Forthcoming Projects Several other projects targeting non-estrogenic compounds Atrazine – 2-D gels 5 Gene expression markers different tissues Androgen indicators Invertebrate sources Mixtures Pulsed exposures Expression Technology Summary Informative Available - microarrays are online or under developed for a number of aquatic species Sensitive Targets changes early in exposure High through-put Making linkages to higher biological levels Assays are being developed for a number of different species representing an array of different ecological catagories (habitat, feeding groups, etc.
